A combination of recombinase polymerase amplification with CRISPR technology rapidly detects goose parvovirus with high accuracy and sensitivity.

Abstract

BACKGROUND: Goose parvovirus (GPV) poses a significant threat to the waterfowl industry, necessitating reliable detection methods. However, conventional techniques are often time-consuming, equipment-dependent, or lack sufficient sensitivity for detecting early-stage infection. In contrast, emerging CRISPR/Cas12a-based systems offer a promising alternative for rapid, sensitive, and on-site diagnostics. METHODS: We developed and optimized a recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay targeting the conservedgene of GPV. The analytical and diagnostic performance of this assay was rigorously validated using plasmid standards and clinical specimens from both experimentally infected and field-collected ducklings. RESULTS: Our developed assay combines RPA with CRISPR/Cas12a technology for rapid GPV nucleic acids detection. This method achieves a detection limit of 10 copies/L of thegene within one hour, demonstrating high sensitivity and rapid turnaround. The assay exhibited exceptional specificity, with no cross-reactivity against other waterfowl viruses, and showed robust reproducibility, with intra- and inter-assay coefficients of variation consistently below 5.0%. Clinical validation using 42 field samples confirmed a diagnostic sensitivity of 100% and 95.5% specificity, showing superior performance to real-time quantitative PCR (qPCR) in both metrics. Furthermore, the assay supports flexible visual readouts using portable blue light transilluminators, facilitating on-site interpretation. CONCLUSIONS: This study established a highly field-deployable RPA-CRISPR/Cas12a assay for rapid, visual detection of GPV with outstanding sensitivity and specificity. Its capability for instrument-free on-site diagnosis via blue light transillumination makes this approach particularly promising for resource-limited settings.